Calcium/calmodulin‐dependent kinase II and nitric oxide synthase 1‐dependent modulation of ryanodine receptors during β‐adrenergic stimulation is restricted to the dyadic cleft

KEY POINTS: The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca(2+)/calmodulin‐dependent kinase II (CaMKII) activation during high‐frequency stimulation. Sympathetic stimulation through β‐adrenergic receptors activates an integrated signalling cascade, enhancing Ca(2+) cycling and is at least partially mediated through CaMKII. Here we report that CaMKII activation during β‐adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs. In contrast, the increase in the Ca(2+) content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca(2+) transient are primarily protein kinase A‐dependent. The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. ABSTRACT: In cardiac myocytes, β‐adrenergic stimulation enhances Ca(2+) cycling through an integrated signalling cascade modulating L‐type Ca(2+) channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca(2+)/calmodulin‐dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca(2+) microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole‐cell voltage‐clamp and confocal line‐scan imaging with Fluo‐4 as a [Ca(2+)](i) indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca(2+) sparks, which are assigned to coupled and non‐coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca(2+) spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces I (CaL) and local Ca(2+) transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca(2+) transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca(2+) content, Ca(2+) transient amplitude and relaxation. In conclusion, CaMKII activation during β‐adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC‐triggered local Ca(2+) release as well as spontaneous diastolic Ca(2+) release whilst PKA is the major pathway increasing global Ca(2+) cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy.